[11] Use of laser capture microdissection for clonal analysis

[11] Use of laser capture microdissection for clonal analysis

[ 11] USE OF LCM FOR CLONALANALYSIS 129 [11] Use of Laser Capture Microdissection for Clonal Analysis By VAL~R1EPARADIS and PIERRE BEDOSSA C l o n ...

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[11] Use of Laser Capture Microdissection for Clonal Analysis By VAL~R1EPARADIS and PIERRE BEDOSSA C l o n a l Analysis: A D i a g n o s t i c Tool i n H u m a n P r o l i f e r a t i o n Clonal study is a useful approach to the analysis of tissue proliferation.l Indeed, clonality helps define the real nature of lesions, resulting in a better understanding of their behavior and appropriate management. Such an assay allows the differentiation between polyclonal and monoclonal lesions. In fact, monoclonal patterns would support neoplastic nature, whether benign or malignant, whereas polyclonality would be consistent with a regenerative or inflammatory process. Thus, clonal studies have been applied mainly to the study of neoplastic transformation and tumor progression. 2-4 The presence of a common and nonrandom genetic alteration in a group of cells confirms their clonal origin only when these particular alterations are acquired early in the neoplastic process and then transmitted to daughter cells. Clonal analysis relies on various tests, including microsatellite analysis (linked or not to the X chromosome) and specific gene rearrangements. 5-7 The choice of test is mainly determined by the nature of the pathologic process of interest. Whereas LOH analysis is a useful tool for the detection of inactivation of tumor suppressor genes, specific gene rearrangements are shown to be useful in the diagnosis of various tumors, especially malignant lymphomas. 7'8 However, some of these genetic modifications may occur late in the course of a neoplastic process, involving only a few subclones. This is avoided in X-linked clonal analysis since the clonal marker investigated in such an approach is constitutively present in each somatic cell independent of the genetic or epigenetic events occuring during neoplastic transformation or clonal expansion. 9 These methods, applied only to informed female patients, based on X chromosome inactivation include the ability to

1 p. j. Fialkow, Biochem. Biophys. Acta 458, 283 (1976). 2 E C. Nowell, Science 194, 23 (1976). 3 B. Vogelstein, E. R. Fearon, S. E. Kern, A. C. Preisinger, M. Leppert, Y. Nakamura, R. White, A. M. Smits, and J. L. Bos, 17. Engl. J. Med. 319, 525 (1988). 4 M. Sternlicht, C. Mirell, S. Safarians, and S. Barsky, Biochem. Biophys. Res. Commun. 199, 511 (1994). 5 T. A. Brentnall, Am. J. Pathol. 147, 561 (1995). 6 j. Koreth, J. J. O'Leary, and J. O. D. McGee, J. Pathol. 178, 239 (1996). 7 K. J. Trainor, M. J. Brisco, J. H. Wan, S. Neoh, S. Grist, and A. A. Morley, Blood78, 192 (1991). 8 A. G. Knudson, Proc. Natl. Acad. Sci. U.S.A. 90, 10914 (1993). 9 B. Vogelstein, E. R. Fearon, S. R. Hamilton, and A. P. Feinberg, Science 227, 642 (1985).


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HUMARAGENE --~Primer 1

Hpa II sites


H Primer 2 eHpalI d i g e s t i ~



~ / / / / ~






m m







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determine the paternally derived X chromosome from the maternally derived one. According to Lyon's hypothesis, genes on either of the two X chromosomes are randomly inactivated by methylation of cytosine residues within promoter regions during early embryogenesis, m Once methylated, such CpG islands are functionally and heritably inactive. It is widely believed that this inactivation is stable, even during the neoplastic change. Therefore, in the context of clonal proliferation, any transformed cell will transmit to its progeny its own X chromosome pattern of inactivation. Consequently, reactive polyclonal processes will have an admixture of cells with inactivated maternal and paternal X chromosomes, whereas clonal processes will have a dominant population of cells with only one inactivated allele. Many genes on the X chromosome are polymorphic and allow the distinction between maternally and paternally inherited X chromosomes. This information is related directly to the frequency of their polymorphism in a population, from 29% heterozygosity for hypoxanthine phosphoribosyltransferase to more than 90% for the human androgen receptor gene (HUMARA). 11 Therefore, most of the studies investigating clonal status examine the methylation status of the HUMARA gene. This gene contains several restriction sites of the methylation-sensitive HhaI and HpalI endonucleases adjacent to a polymorphic [(CAG)n] repeat region in exon 1. Enzymatic digestion with HhaI or HpalI, followed by PCR targeted to the HUMARA gene region, using primers whose product spans both the HpalI sites and the [(CAG)n] polymorphism, will result in amplification of the methylated undigested alleles but not the unmethylated alleles (Fig. 1). Variations in the lengths of the [(CAG)n] repeats on the patemal and maternal X chromosomes will yield HUMARA alleles of different lengths which can be resolved by gel electrophoresis. LCM a n d C l o n a l A n a l y s i s Clonal analysis appears to be an accurate tool in the understanding and management of various human disorders. However, reliable interpretation of the results of clonality tests depends mainly on correlation with tissue morphology. In that ~0 M. E Lyon, Biol. Rev. 47, 1 (1972). ll D. G. Gilliland, K. L. Blanchard, J. Levy, S. Perrin, and H. E Bunn, Proc. Natl. Acad. Sci. U.S.A. 88, 6848 (1991).

FIG. 1. Top: Structure of the androgen receptor gene (exon 1). A highly polymorphic CAG repeat sequence located downstream of a potentially methylated CG island allows a reliable yield of both components in a single chain reaction amplification. Both alleles, easily differentiated by size, can be amplified only from the inactive X chromosome (Xi) in the hpalI-digested sample. Bottom: Allelic patterns of androgen receptor gene in monoclonal and polyclonal proliferations. The presence of both alleles in hpalI-digested samples (D) characterizes polyclonal proliferation (left), whereas the presence of a single allele in hpalI-digested samples (D) [two alleles in undigested samples (U)] defines monoclonal proliferations (right).



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context, the use of LCM provides major benefits in the clonal evaluation since it allows accurate selection of the cell population of interest. As a matter of fact, both normal and lesional tissues are composed of a mixture of cell types, such as epithelial, mesenchymal, endothelial, and inflammatory cells. This is obvious, especially in neoplasia where the neoplastic cells are intimately associated with the nonneoplastic stromal component. Such cell heterogeneity could lead to false polyclonal results in the analysis of the clonal status of a lesion. It is known that contaminating stromal and inflammatory cells within tumors should not exceed 20% in LOH studies, and that an increase in sensitivity of more than 50% in allelic imbalance analysis is obtained by using microdissected cell populations compared with crushed frozen tumor samples. 12 It appears, then, that isolation and enrichment of defined cell populations from biological samples are necessary steps for improving the sensitivity of the clonal analysis. The search for such higher sensitivity prompted the development of new methods of tissue microdissection. The early assisted-microdissection techniques, involving manual or micromanipulator guidance of a needle to scrape off the cells of interest under a microscope, were in fact not accurate enough to isolate cells of interest in a cellular mixture.13,14 They are now replaced by technologies based on laser capture. Two main procedures are currently available: one using a pulsed ultraviolet laser with a small beam focus to cut areas of cells of interest by photoablation of adjacent tissue; the other based on the selective adherence of visually targeted cells to a thermoplastic membrane activated by a low energy infrared laser pulse. 15-18 This latter microdissection technique is attractive since it does not destroy adjacent tissues of the area of interest and allows sequential samplings of the same slide. Whichever system is used, collections of pure cell populations in a simple and quick manner are now easily accomplished in a one-step transfer. Such collections are also allowed by the good preservation of morphology of both captured cells and the residual tissue. A p p l i c a t i o n s of LCM a n d C l o n a l A n a l y s i s in H u m a n D i s o r d e r s Use of LCM in clonal analysis gave rise to significant advances in the understanding of the pathogenesis of various human diseases, especially those of 12 H. E. Giercksky, L. Thorstensen, H. Qvist, J. M. Nesland, and R. A. Lothe, Diagn. Mol. Pathol. 6, 318 (1997). 13 M. Emmert-Buck, M. J. Roth, Z. Zhuang, E. Campo, J. Rozhin, B. E Sloane, L. A. Liotta, and W. G. Stetler-Stevenson, Am. J. Pathol. 145, 1285 (1994). 14 S. Noguchi, H. Motomura, H. Inaji, S. Imaoka, and H. Koyama, Cancer Res. 54, 1849 (1994). 15 I. Becker, K. F Becker, M. H. R6hrl, G. Minkus, K. Schiitze, and H. Htifler, Lab. Invest. 75, 801 (1997). 16 M. BiShm, I. Wieland, and K. Schiitze, Am. J. Pathol. 151, 63 (1997). 17 M. R. Ernmert-Buck, R. E Bonner, P. D. Smith, R. E Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 18 R. E Bonner, M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997).

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uncertain behavior. It gives further insights into human diseases the morphologic patterns of which do not allow per se the distinction between a reactive and neoplastic lesion. For instance, whereas initial studies demonstrated the neoplastic condition of Langerhans' cell histiocytosis, recent results showed that pulmonary Langerhans' cell histiocytosis nodules are polyclonal, suggesting that LCH involving specific sites, such as lungs, might behave rather as regenerative disorders despite some systemic forms of the disease which are clonal. 19'2° Such an approach also clarifies the nature of several entities usually considered neoplastic. For instance, the analysis of the epithelial component of Warthin's tumor, collected by microdissection, showed patterns of polyclonal proliferation in the H U M A R A assay, suggestive of a nonneoplastic tumor-like condition. 21 In addition, clonal analysis of morphologically distinct components within a tumor, successfully performed with the use of LCM, provided further highlights on the cellular origin in tumors and the relationships between all components. This has been illustrated in cases of pleomorphic adenomas of salivary glands which displayed a monoclonal pattern, showing that the two morphologically different areas in these tumors, the stromal and the epithelial components, arise from the same clone. 22 Such an approach has also been very useful in the study of liver cirrhosis which constitutes the main preneoplastic change in the liver. Indeed, the detection of the minute potential premalignant precancerous monoclonal nodules is critically important, helping in the management of patients with chronic liver diseases and cirrhosis. Before the development of LCM, clonal analysis was performed on large nodules, also called macronodules. Such analysis demonstrated that aproximately 50% of them were monoclonal in origin, suggestive of neoplasia. 23'24 Interestingly, clonal status did not correlate with the morphological classification of the macronodules, indicating the poor prognostic value of pathological classifications. 24 These results prompted us to extend this study to the analysis of smaller cirrhotic micronodules. 25 To address this issue, liver micronodules of a mean size of 1 mm were microdissected from one 16-/zm serial paraffin section of cirrhotic tissue by LCM with a PixCell instrument (Arcturus Engineering, Mountain View, CA). Parameters of the LCM used included a laser diameter of 30/xm, a laser power of 50 mW, and a pulse length of 10 ms. Each micronodule was microdissected separately after being identified in the adjacent hematoxylin and 19C. L. Willman, L. Busque, B. B. Griffith, B. E. Favara, K. L. McClain, M. H. Duncan, and D. G. Gilliland, N. Engl. J. Med. 33L 154 (1994). 20S. A. Yousem,T. V. Colby, Y.-Y.Chen, W.-G. Chen, and L. M. Weiss,Am. J. Surg. Pathol. 25, 630 (2001). 21 K. Honda, K. Kashima, T. Daa, S. Yokoyama,and I. Nakayama, Hum. Pathol. 31, 1377 (2000). 22P.-S. Lee, M. Sabbath-Solitare, T. C. Redondo, and E. H. Ongcapin,Hum. Pathol. 31,498 (2000). 23Z. Aihara, S. Noguchi, Y. Sasaki, H. Nakano, and S. Imaoka, Gastroenterology 107, 1805 (1994). 24V. Paradis, I. Laurendeau, M. Vidaud, and E Bedossa, Hepatology 28, 953 (1998). 25V. Paradis, D. Darg~re, E Bonvoust, L. Rubbia-Brandt, N. Ba, E Bioulac-Sage, M. Vidaud, and E Bedossa, Lab. Invest. 80, 1553 (2000).



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FIG, 2. Distribution of mono- and polyclonal micronodules microdissected from liver cirrhosis. Clonal analysis showed a random distribution of mono- (M) and polyclonal (P) micronodules in a case of cirrhosis without macronodule.

eosin stained section. The captured micronodular tissue was immediately placed in a 0.5-ml microfuge tube containing proteinase K. The HUMARA assay was performed after DNA extraction. In this study, 51% of the micronodules were monoclonal with a random distibution throughout the liver cirrhosis, as shown in Fig. 2. These results demonstrate the presence of neoplastic nodules in liver cirrhosis and suggest that monoclonal nodules could represent potential markers for the future development of a carcinoma.

Specific A d v a n c e s t h r o u g h t h e U s e o f LCM A s s o c i a t e d with Clonal Analysis Clonal analysis combined with LCM technology also provides further highlights on specific issues. For instance, before the development of assisted microdissection techniques, tumor heterogeneity was poorly demonstrated at the molecular level, although it is a common feature of human malignancies, well-recognized by histological analysis. Study of the adjacent loci of cells exhibiting various morphological patterns included inside a lesion is now feasible with the use of LCM. This has been illustrated in the analysis of the molecular clonality of melanomas. Using LOH microsatellite markers after LCM, authors assessed intratumor heterogeneity




in about 7% of melanoma cases. 26 Intratumoral molecular heterogeneity was also described in aflatoxin-induced murine lung tumors based on p53 status. 27 Some true neoplastic disorders have been found to be oligoclonal. Such findings have been demonstrated by the use of careful microdissection as illustrated by Cabras e t al. 28 in a study investigating the clonality of morphogically distinct areas of gastric lymphomas. This study clearly showed that some gastric lymphomas exhibited biclonal IgH genes, consistent with the presence of two malignant clones arising from different progenitor cells. At last, the combination of clonal analysis and LCM could provide further evidence for the nature of field cancerization as a discontinous, multifocal, and polyclonal process, as has been proposed in some tissues. 29'3° These data confirm the role of LCM in the study of defined cell components within the lesion, especially since studies are carried out on very few cells. In this respect, several papers reported that clonality can be assessed when clonally derived cells comprise 20% or more of the population. 19, 31,32 Pitfalls a n d D r a w b a c k s The application of laser-assisted microdissection techniques can be widespread, to DNA, RNA, and protein analysis, since it has been shown that these procedures do not significantly alter additional molecular analysis. However, integrity and quality of the biological material provided should be checked, since these techniques can be applied on both frozen and archival tissue specimens Indeed, the main parameter influencing clonal analysis is specimen fixation, which critically determines the conservation and integrity of the nucleic acids. For instance, fixation leads to some degree of fragmentation of DNA and RNA and induces several chemical modifications, including cross-linking between amino groups on DNA bases, resulting in potentially poor PCR amplification. Appropriate controls, able to check the quality of the material and the efficiency of the molecular procedures, should be simultaneously run. Finally, clonal analysis can be quite easily applied to archival tissue specimens when analysis is performed on samples fixed without 26 T. Nakayama, B. Taback, R. Turner, D. L. Morton, and D. S. B. Hoon, Am. J. Pathol. 158, 1371

(2001). 27 A. S. Tam, J. E Foley, T. R. Devereux, R. R. Maronpot, and T. E. Massey, Cancer Res. 59, 3634 (1999). 28 A. D. Cabras, S. Candidus, E Fend, M. Kremer, S. Schulz, C. Bordi, G. Weirich, H. Hrfler, and M. Werner, Lab. Invest. 81, 961 (2001). 29 H. K. Yang, R. I. Linnola, N. K. Conrad, M. J. Krasna, S. C. Aisner, B. E. Jonson, and M. J. Kelley, Int. J. Cancer 64, 229 (1995). 3o R. D. Mashal, M. L. S. Fezjo, A. J. Friedman, N. Mitchner, R. A. Nowak, M. S. Rein, C. C. Morton, and J. Sklar, Genes Chromosomes Cancer 11, 11 (1994). 31T. Enomoto, T. Haba, M. Fujita, T. Hamada, K. Yoshino, R. Nakashima, H. Wada, H. Kurachi, K. Wakasa, M. Sakurai, Y. Murata, and K. R. Shroyer, Int. J. Cancer 73, 339 (1997). 32 K. Krohn, D. Fuhrer, H. E Holzappel, and R. Paschke, J. Clin. EndocrinoL Metab. 83, 130 (1998).



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fixatives containing picric acid. T M The possibility of performing such studies on archival tissue has led to further applications in the field of oncology where clonal analysis of microscopic premalignant lesions and multifocal small cancers is inevitably discussed. Numerous situations have been investigated, such as cervical intraepithelial neoplasia 31,33 or colonic neoplasia. 34 This type of analysis, usually with very tiny samples, introduces the concept of patch size. 35 A patch is usually regarded as a group of cells which share a common genotype and have the same X chromosome inactivation pattern. Regarding this concept, the sample size becomes a critical factor which should be taken into account in studies investigating clonality. Therefore, this notion of patch size can impair clonal analysis in tissue samples that are too small. To date, there have been few data concerning patch size in human tissues. However, a large discrepancy in patch size can be observed in tissues from various origins, from a few mlTl 2 in the myometrium or the liver to cm 2 in the bladder or arteries. 36-39 It is then assumed that any tumor arising within a patch will be, by definition, monoclonal with an X-linked marker. Consequently, X chromosome inactivation cannot be relied on to assess clonality because of the very large patch size of tissue in some cases, such as arteries. An additional potential drawback concerns skewed-X chromosome expression. This phenomenon of extreme lyonization can also mimic clonal derivation of a group of cells. This occurs when there is skewing in the random inactivation of the X chromosome during embryogenesis which may vary from tissue to tissue. Such skewed profiles are transmitted to all somatic cells of normal tissue, resulting in a false monoclonal pattern. To optimally control for lyonization, it is necessary to study control normal tissues, expected polyclonal, that are closely related to the tissue of interest. In conclusion, molecular techniques such as PCR-based methods using X-linked genes as clonal markers have been increasingly used to demonstrate the clonal nature of many tumoral processes. Finally, the development of LCM importantly contributed to a broader application in various human disorders allowing a better knowledge of their biological behavior.

33 H.-S. Park, R. A. Goodlad, and N. A. Wright, Am. J. Pathol. 147, 1416 (1995). 34 M. R. Novelli, J. A. Williamson, I. P. M. Tomlinson, G. Elia, S. V. Hodgson, I. C. Talbot, W. E Bodmer, and N. A. Wright, Science 272, 1187 (1996). 35 G. H. Schmidt and R. Mead, BioEssays 12, 37 (1990). 36 G. Linder and S. M. Gartler, Science 150, 67 (1965). 37 T. Ochiai, Y. Urata, T. Yamano, H. Yamagishi, and T. Ashihara, Hepatology 31, 615 (2000). 38 y. C. Tsal, A. R. Simoneau, C. H. Spruck, P. W. Nichols, K. Steven, J. D. Buckley, and P. A. Jones, J. UroL 153, 1697 (1995). 39 I.-M. Chung, S. M. Schwartz, and C. E. Murry, Am. J. Pathol. 152, 913 (1998).